The sense of taste is important in food selection and enjoyment. Inability to taste can result in poor nutrition and a lower quality of life. Although important advances in the understanding of taste stimulus transduction have been made, relatively little is known about the molecular mechanisms that regulate the observed physiological responses in taste receptor cells. The principal investigator has previously cloned a novel and functional potassium channel (Kv2.3). The specific aims of this application are: Aim 1. Determine distribution of expression of fish Kv2.3 mRNA in the tissues of catfish. The distribution of mRNA expression in the barbel (taste tissue) will be determined by in situ hybridization using the two specific non-overlapping RNA probes. The size and distribution of the K+ channel mRNA in the catfish will be determined by Northern blot or Rnase protection analysis. Aim 2. Determine molecular mass of recombinant and native Kv2.3 proteins. Determine which cells in taste buds express Kv2.3. Peptides corresponding to the highly antigenic sequences of the Kv2.3 polypeptide will be synthesized. Polyclonal anti-peptide antibodies will be produced. Mass and extent of glycosylation of native and recombinant Kv2.3 will be determined by immunoblot analysis. Aim 3. Characterize the ATP-mediated modulation of Kv2.3 channel function. Pharmacologically, electrophysiologically, and biochemically separate or distinguish the rapidly inactivating current component from the sustained component. ATP dependence, kinetic properties, and pharmacology will be determined for each component before and after depolymerization of actin by two-electrode voltage of patch clamp analysis of Kv2.3 channels expressed in Xenopus oocytes and human IM9 b-lymphocytes. Aim 4. Characterize modulation of Kv2.3 channel activity by phosphorylation, and identify which sites are phosphorylated. Kv2.3 channel activity will be measured with specific activators and inhibitors of kinases and phosphatases. Phosphorylated residues will be identified by two-dimensional phosphopeptide analysis and coincident phosphoamino acid analysis. Polyclonal antibodies to specific phosphorylated residues will be produced, and used to determine whether the same sites in the sequence of the native protein are phosphorylated in vivo or in slices.